Breast cancer is a significant health problem for women in the United States and throughout the world. Despite recent advances in detection and treatment of the disease, breast cancer remains the second leading cause of cancer-related deaths in women. Management of the disease currently relies on a combination of early diagnosis through routine breast screening procedures and aggressive treatment. Such treatment may include surgery, radiotherapy, chemotherapy, hormone therapy or combinations of these therapies.
Ninety-five percent of all breast tumors, at least initially, are dependent on estrogens for growth. Estrogens are steroid hormones that are essential for normal sexual development and functioning of female reproductive organs. Estrogens are also important for growth, differentiation, and functioning of the testis, epididymis and prostate in males. Estrogens also have important non-reproductive effects on bones and the heart. Estrogens comprise a group of natural and synthetic substances. Natural estrogens include estradiol (i.e., 17-xcex2-estradiol or E2), estrone and estriol. Estrogens are sometimes given therapeutically in the form of a conjugate, such as for example, ethinyl estradiol, conjugated estrogens or diethylstilbestrol.
Tissues in the body that are responsive to estrogens are called xe2x80x9cestrogen-sensitivexe2x80x9d or xe2x80x9cestrogen-responsivexe2x80x9d tissues and include cells of the urogenital tract, cardiovascular system and skeletal system. The cells that comprise estrogen-sensitive tissues contain estrogen receptors (ER). ER can be of the xcex1 type or xcex2 type. Estrogens enter cells and bind to ER in the cytoplasm of such cells and an estrogen-ER complex is formed. Herein, a molecule such as estrogen that binds to a receptor is generally called a xe2x80x9cligand.xe2x80x9d Herein, a receptor such as ER that has formed a complex with a ligand is called a xe2x80x9cligandedxe2x80x9d receptor.
Once the estrogen ligand binds to ER, the estrogen-ER complex migrates to the nucleus of the cell and binds to specific sequences of DNA within the cellular genome called xe2x80x9cestrogen response elements.xe2x80x9d Such estrogen response elements are located in the promoters of specific genes in the cell nucleus.
Binding of the estrogen-ER complex to estrogen-responsive elements causes activation or suppression of the transcription of the specific genes (Beato, et al., 1995, Cell, 83:851-7.; Katzenellenbogen, et al., 1995, J Steroid Biochem Mol Biol, 53:387-93.; Tsai and O""Malley, 1994, Annu Rev Biochem, 63:451-86.). The activation or suppression of specific gene transcription is one type of molecular and/or cellular response that can result from formation of a ligand-receptor complex. When such a response occurs, the receptor is said to have been xe2x80x9cactivated.xe2x80x9d
Estrogen-ER complexes, therefore, act as transcription factors to regulate the expression of these genes. When a ligand binds to a receptor and a molecular and/or cellular response (e.g., transcriptional regulation of genes) occurs, such ligands are referred to as xe2x80x9cagonistsxe2x80x9d and the response produced is called xe2x80x9cagonism.xe2x80x9d Herein, therefore, the term agonist refers to ligands, such as estrogen, that produce the molecular and/or cellular responses.
Estrogens and ER play significant roles in certain human cancers, breast cancer being one specific example. Cells in female breast tissue normally contain ER. Interaction of estrogens with ER in breast cells normally causes the breasts to grow at puberty and again during pregnancy. Since breast cells normally contain ER, it is not surprising that cells comprising tumors of the breast also contain ER. Ninety-five percent of all breast tumors, at least initially, have ER and are dependent on estrogens for growth. In such breast tumor cells, estrogens acting via the ER, dramatically escalate proliferative and metastatic activity (Osborne, et al., 1980, Cancer, 46:2884-8.).
Treatment of such ER-positive breast tumors comprises administration to the individual with the tumor, compounds such as tamoxifen (TOT). TOT can also administered to individuals who may be at high risk for developing breast tumors in the future, for the purpose of prevention of such tumors. Chemically, tamoxifen is one of a number of compounds referred to as triphenyethylene derivatives. Tamoxifen is a mainstay of breast cancer treatment and inhibits the proliferation promoting effect of estrogens (Katzenellenbogen, et al., 1995, J Steroid Biochem Mol Biol, 53:387-93.; Osborne, et al., 1980, Cancer, 46:2884-8.; Jordan and Murphy, 1990, Endocr Rev, 11:578-610.). Like estrogens, TOT binds to ER and, therefore, is also an ER ligand. Unlike estrogen binding to ER, however, TOT binding to ER does not result in production of significant molecular and/or cellular responses. The changes in gene expression resulting from TOT binding to ER are significantly less in magnitude than those resulting from estrogen binding to ER. Such decreased responses are referred to as xe2x80x9cpartial agonism.xe2x80x9d Ligands such as TOT, that result in partial agonism, are referred to as xe2x80x9cpartial agonists.xe2x80x9d
Of significance is that binding of ER by TOT prevents estrogens from producing their effect on ER (i.e., the partial agonist precludes effects of the agonist). Since estrogens are prevented from producing a molecular and/or cellular response through the ER, the response produced in the presence of both estrogens and TOT will be partial agonism, rather than agonism. Such partial agonism is the basis by which TOT impairs breast tumor growth (i.e., by blocking the agonist effects of estrogens).
With regard to TOT, while it is effective in preventing proliferation of ER-positive breast tumor cells (i.e., cells that contain ER) in the early stages of breast cancer treatment, such ER-positive tumor cells invariably develop resistance to TOT. That is, after a time (e.g., 5 years), TOT is no longer effective in preventing estrogen stimulation of tumor proliferation and, in fact, causes stimulation of proliferation of ER-positive tumor cells.
The high mortality observed in breast cancer patients indicates that additional methods and tools for diagnosing and treating breast cancer are needed. Methods and tools for differentiating between normal breast tissue and cells and cancerous breast tissue and cells are desirable. Additional methods and tools for reducing or inhibiting the growth or proliferation of breast cancer cells are also desirable.
The present invention provides tools and methods for differentiating normal breast tissue and cells from cancerous breast tissue and cells. The tools are derived from a novel tumor suppressor gene designated as Estrogen Downregulated Gene (EDG1) that is down-regulated by estrogen in mammary epithelial cells. EDG1 encodes a protein referred to hereinafter as the xe2x80x9cEDG1xe2x80x9d protein (SEQ. ID. NO.2). In one aspect the tool is an isolated polynucleotide which encodes the EDG1 protein. In one embodiment, the isolated polynucleotide comprises the nucleotide sequence of SEQ ID NO.1. The present invention also relates to fragments of the isolated polynucleotide that can be used as probes or primers for identifying cells that are or are not expressing EDG1.
In another aspect, the tool is a monoclonal antibody which is immunospecific for the EDG1 protein. The antibody may further comprise a detectable label, such as a fluorescent label, a chemiluminescent label, a radiolabel or an enzyme. Also encompassed are hybridoma cells and cell lines that produce such antibody. In another aspect, the tool is a polyclonal sera, antibodies of which bind immunologically to the EDG1 protein.
In another aspect, the present invention provides a method of detecting cancerous cells in an hormone responsive tissue test sample. Preferably, the sample is a prostate tissue, ovarian tissue, testes tissue, uterine tissue, cervical tissue or, more preferably a breast tissue sample. In one embodiment, the method comprises contacting the sample or a protein extract therefrom with at least one antibody to the EDG1 protein under conditions wherein antibody binding to the EDG1 protein occurs; and assaying for the presence or absence of a complex between the antibody and a protein in the sample, wherein a decrease in the level of the antigen-antibody complex, as compared to the levels found in a sample of control cells, indicates that the sample comprises cancerous cells. Preferably, the assay is an immunocytochemical assay which permits determination of the intracellular location of the antigen-antibody complexes. In another embodiment, the method comprises assaying for the presence of EDG1 transcript in the sample, wherein a decrease in the level of the EDG1 transcript in the sample, as compared to the level of the EDG1 transcript in a control sample, denotes that the test sample comprises cancerous cells.
The present invention also relates to the protein encoded by EDG1 and biologically active or immunologically reactive fragments thereof. In one embodiment the EDG1 protein has the amino acid sequence of SEQ ID NO.2. In one embodiment the EDG1 protein fragment has the amino acid sequence of SEQ ID NO.3.
The present invention also provides a method for decreasing proliferation of breast cancer cells, prostate cancer cells, testicular cancer cells, and ovarian cancer cells. Such method comprises increasing levels of the EDG1 protein in such cells. In one embodiment, the cells are contacted with the EDG1 protein or a biologically active equivalent or fragment thereof under conditions permitting uptake of the protein or fragment. In another embodiment, the cells are contacted with (i) a nucleic acid encoding the EDG1 protein, and (ii) a promoter active in the cancer cell, wherein the promoter is operably linked to the region encoding the EDG1 protein, under conditions permitting the uptake of the nucleic acid by the cancer cell. The cancer cell may be derived from an endocrine tissue such as breast, ovary, prostate or testes tissue.
The present invention also provides a method for inhibiting the transcriptional activity of estrogen-liganded ERxcex1 in cancer cells. Such method comprises increasing the levels of the EDG1 protein in such cells.